Mechanistic insights into the interaction between the host gut microbiome and malaria

Malaria is a devastating infectious disease and significant global health burden caused by the bite of a Plasmodium-infected female Anopheles mosquito. Gut microbiota was recently discovered as a risk factor of severe malaria. This review entails the recent advances on the impact of gut microbiota composition on malaria severity and consequence of malaria infection on gut microbiota in mammalian hosts. Additionally, this review provides mechanistic insight into interactions that might occur between gut microbiota and host immunity which in turn can modulate malaria severity. Finally, approaches to modulate gut microbiota composition are discussed. We anticipate this review will facilitate novel hypotheses to move the malaria-gut microbiome field forward.


Introduction
Malaria is an infectious disease caused by the bite of a female Anopheles mosquito infected with the parasite Plasmodium.Malaria remains a significant burden on the global healthcare system, causing more than 627,000 deaths and 241 million cases in 2020 [1,2].Greater than 90% of infections and severe malaria in humans is caused by P. falciparum, with additional infections caused by other Plasmodium species including P. vivax, P. malaria, P. ovale, and P. knowlesi [3].Sequestration of infected RBCs in internal organs lead to widespread organ damage and is a major cause of death in patients with severe P. falciparum malaria [4,5].However, additional factors that determine the severity of malaria in humans are still evolving.Recently, it was shown that the gut microbiome (i.e., microorganism including their genetic content, microbial products, and environment within the gut) is a risk factor of severe malaria [6,7].
The phrase "you are what you eat" which can be extended to "what you eat is your gut microbiome" is very true in the context of gut microbiome that play a critical role in health and disease of individuals [8][9][10].Bacteria make up the major organic fraction of feces (approximately 25% to 54% of dry solids) in humans [11].Approximately 70% to 80% of immune cells are located in the gut that are trained by the intestinal microbiome [12].Innate and adaptive immunity are required to control Plasmodium infection.Thus, gut microbiota that influence local and systemic immune system have the potential to significantly impact antimalarial immunity [13][14][15][16][17].
Here, we have provided mechanistic insight into the role of gut microbiome in shaping malaria severity, including severe malaria anemia (SMA) and cerebral malaria.The presence of specific gut bacteria like Ruminococcaceae, Lachnospiraceae, Bacteroides, and Blautia were associated with severe malaria while Bifidobacterium and Escherichia are correlated with better outcome following Plasmodium infection.In addition to preinfection bacteria composition correlating with malaria outcomes, bacteria populations have also been shown to change in abundance following infection.Bacterial genera including Bacteroides, Alistipes, and Clostridia are relatively increased while Ruminococcaceae, Prevotellaceae, and Ruminococcus are decreased in abundance during Plasmodium infection.Importantly, gut bacteria represent a druggable target to boost antimalarial immunity and decrease malaria severity.

Associations between human gut microbiome and malaria
In 2014, Yilmaz and colleagues showed that older individuals (4 to 25 years old) with higher levels of Galα1-3Galβ1-4GlcNAc-R (α-gal) glycan-specific IgM antibodies in plasma were significantly protected against P. falciparum infection [18].However, younger children (3 months to 4 years old) had no association between protection from P. falciparum infection and α-gal IgM antibodies in the plasma.Evolutionarily, humans do not express α-gal, allowing them to generate anti-α-gal antibodies when colonized by α-gal expressing bacteria [19].Other microorganisms, including Plasmodium, express α-gal, providing an opportunity for cross-reaction between bacterial-induced anti-α-gal antibodies and other microbes.The authors further confirmed the role of α-gal produced by Escherichia coli O86:B7, a member of gut microbiota, in the mouse model of malaria (Fig 1).Anti-α-gal IgM antibodies confer protection against Plasmodium sporozoite infection independent of complement induced polymorphonuclear leukocyte recruitment, but rather by complement-induced cytotoxicity.Anti-α-gal IgM antibodies also conferred protection, in part, via blocking sporozoite migration and infection of hepatocytes, thereby blocking development of exoerythrocytic forms within hepatocytes.In contrast, anti-α-gal antibodies had no effect on the erythrocytic stage of infection in mice [18].Additionally, in an RTS,S/AS0 vaccination trial conducted in Mozambique and Ghana, vaccinated infants (1.5 to 3 months) who had higher level of anti-α-gal IgM plasma antibodies were protected against clinical malaria over a one-year follow up compared to infants with low levels of IgM plasma antibody [20].Nonetheless, anti α-gal IgM levels were not associated with protection against malaria in vaccinated older children (5 to 17 months) [20].
A study by Yooseph and colleagues in 2015 [28] (using the same prospective cohort of Malian children as used by Yilmaz and colleagues (2014) [18]) reported that younger children (average age of 1.4 years) had a delayed time to first P. falciparum infection with median 121 days (95% CI 101 to 150) compared to older children (average age of 9.1 years) with median 85 days (95% CI 73 to 99).By accounting for age, gender, anemia, HbAS, S. hematobium infection, splenomegaly, and distance to river, the authors concluded that younger children stool microbiota significantly protected against prospective risk of P. falciparum infection compared to stool microbiome in older children.However, stool microbiota did not correlate with protection against febrile malaria episodes between the young and old children.Older children with increased prospective risk and shorter time to P. falciparum infection had significantly higher abundance of Ruminococcaceae unclassified, Lachnospiraceae unclassified, Prevotellaceae unclassified at family level; and Faecalibacterium, Blautia, and Dorea at genus level compared to younger children.Young children with delayed P. falciparum infection had significantly higher relative abundance of Bifidobacterium, Streptococcus, Escherichia/Shigella compared to older children among others (Fig 1).It was also reported that the stool microbiota composition was different between children (average age 3.2 years) who had a persistent asymptomatic P. falciparum infection carried over from the previous malaria transmission season than children (average age 1.3 years) who had no P. falciparum infection at the end of sixmonth dry season before start of subsequent six-month malaria season in Mali.This result Gut bacteria associated with human malaria.To date 5 peer-reviewed studies have been published on the impact of the gut microbiota in Plasmodium infection in humans.A cohort of children in Kalifabougou, Mali was used for 2 prospective studies (Yilmaz and colleagues (2014) [18] and Yooseph and colleagues (2015) [28]).Additionally, 3 case-control studies investigated association of gut microbiota at the time of Plasmodium infection (Huwe and colleagues (2019) [32] and Easton and colleagues (2020) [33]) and SMA (Mandal and colleagues (2021) [6]).Prospective and case-control studies have their own advantages and disadvantages.Bacteria associated with the clinical outcome is shown.Pf: P. falciparum, neg: negative, pos: positive.may imply that gut microbiota composition in dry season might be involved in persistence or asymptomatic P. falciparum infection.
A limitation of Yilmaz and colleagues (2014) [18] towards our understanding into how gut microbiota impact Plasmodium infections and severity of malaria is that the authors did not identify the source of anti-α-gal IgM antibodies in the participants or if the abundance of E. coli O86:B7 in stool is associated with protection against P. falciparum infection.Likewise, Yooseph and colleagues (2015) [28] did not account for baseline anti-α-gal IgM antibody levels or if they are associated with time to delayed P. falciparum infection in younger kids during gut microbiota analysis.Thus, it's not clear if the delayed P. falciparum infection in younger kids (1.4 years) compared to older kids is due to cross-reactivity between anti-α-gal IgM antibodies and P. falciparum or with differential gut microbiota composition.Noteworthy, Yooseph and colleagues (2015) [28] reported higher abundance of Escherichia/Shigella in the stool of younger kids with delayed protection from P. falciparum infection.Additionally, in the same Malian cohort P. falciparum reticulocyte-binding protein homologue 5 (PfRH5) specific IgG antibody was associated with a longer time to blood-stage infection and first febrile malaria and enhanced p53 expression in monocytes was predicted to be protective against febrile malaria [29,30].Finally, P. falciparum Schizont Egress Antigen-1 anti-(PfSEA-1) in plasma is associated with a decreased risk of severe malaria in 1.5 to 4 years old kids in a holoendemic area of Tanzania [31].These observations highlight the complexity of human malaria outcomes and the challenge to account for these while assessing the contribution of gut microbiota as a risk factor.
A study by Huwe and colleagues (2019) [32] in India where soil-transmitted helminths and malaria are endemic, reported a significantly higher abundance of Lactobacillus genus in the stool of individuals aged 0 to 68 years old infected with P. falciparum and P. vivax compared to noninfected individuals (Fig 1).Within this cohort (n = 68), 46% were infected with Plasmodium [32].In another study, Easton and colleagues (2020) [33] found that stool bacterial taxa of Colombian children aged 4 to 16 years were stronger predictors of P. vivax parasitemia levels compared to the host peripheral blood transcriptome response or complete blood count.Although principal component analysis showed that overall gut microbiota composition was not significantly different between P. vivax-infected and uninfected children, differential abundance analysis identified higher prevalence of stool Bacteroides and lower abundance of Prevotella and Clostridiaceae were associated with P. vivax infection compared to uninfected individuals (Fig 1 ).A critical limitation of these studies is the comparison of gut bacteria between Plasmodium-infected and uninfected children because it is not possible to know what the malaria outcomes would be in the uninfected children if they, too, were infected with Plasmodium.This limitation is addressed in a study that compared gut bacteria between Ugandan children (0.5 to 4 years old) with an asymptomatic P. falciparum infection to children with severe malarial anemia (SMA) [6].Children with SMA had significantly different gut bacteria composition compared to children that had asymptomatic P. falciparum infection.Higher abundance of stool bacteria including Escherichia coli, Parabacteroides distasonis, Bacteroides caccae, and Klebsiella pneumoniae among others were predictive of SMA (Fig 1).The possible mechanism by which gut bacteria may mediate immunity to malaria is described in later section.Limitations of this study include the small sample size (n = 7) of the Ugandan children with asymptomatic P. falciparum infection and the use of case-controls as opposed to a longitudinal prospective study.The latter limitation is potentially important as P. falciparum may cause changes in gut bacteria (discussed below).

Host gut microbiome impacts pathogenesis of malaria-Lessons from nonhuman vertebrate models
Several publications have reported the association between gut microbiota composition and malaria outcomes in mouse malaria models.Since 2016, it has been shown in several studies that baseline gut bacteria structure, function, and composition dictate susceptibility to P. yoelii 17XNL hyperparasitemia in specific-pathogen-free (SPF) C57BL/6 and BALB/c mice from different vendors and isolated barrier units (IBUs) [6,[34][35][36][37][38][39].These outcomes are not restricted to mice from different vendors and IBUs, as antibiotic-induced changes in gut microbiota, both before P. yoelii 17XNL and up to 7 days after P. yoelii 17XNL infection can impact severity of malaria [6].Additionally, in outbred Swiss Webster mice, gut microbiota composition determined P. chabudi chabudi AS infection pregnancy malaria outcomes [40].Gut microbiota is a consortium of bacteria, fungi, archaea, viruses, and helminths.Among these microbes, it was shown that gut bacteria modulate the severity of P. yoelii 17XNL hyperparasitemia [6].However, the potential contribution of other members of gut microbiota (e.g., fungi, archaea, and helminth) cannot be overlooked.Indeed, intestinal helminths are known to modulate malaria outcomes mostly exacerbating P. falciparum and P. vivax infection, yet the effects of intestinal helminths on malaria outcomes remains controversial [33,[41][42][43][44].
Gut bacteria could exert an effect on malaria outcomes by providing protection from severe malaria or by causing susceptibility to severe malaria.Presently, results from the P. yoelii 17XNL hyperparasitemia model suggest bacteria cause susceptibility, rather than resistance, to P. yoelii 17XNL hyperparasitemia.First, P. yoelii 17XNL hyperparasitemia-susceptible C57BL/ 6N mice treated with antibiotics have low parasitemia, while antibiotic-treated hyperparasitemia-resistant C57BL/6N mice were largely unaffected [6].Second, fecal microbiota transplant (FMT) from hyperparasitemia-susceptible C57BL/6N mice➔hyperparasitemia-resistant C57BL/6N mice confers susceptibility while hyperparasitemia-resistant FMT➔hyperparasitemia-susceptible mice did not confer resistance [6].Although these results favor gut bacteria can cause susceptibility to P. yoelii 17XNL hyperparasitemia, additional research in this model, and others, may identify protective roles of bacteria against severe malaria.C57BL/6N mice that are susceptible to severe hyperparasitemia have decreased numbers of germinal center (GC) B cells and follicular helper T (Tfh) cells and decreased titers of P. yoelii 17XNL-specific antibodies that recognize a smaller repertoire of P. yoelii 17XNL antigens [6,34].C57BL/6N mice that develop P. yoelii 17XNL hyperparasitemia have significantly higher abundance of gut bacteria including Bacteroidaceae, Prevotellaceae, and Sutterellaceae at family level and Clostridium papyrosolvens, Alistipes putredinis, and Alistipes timonensis species compared to C57BL/6N mice resistant to P. yoelii 17XNL hyperparasitemia (Fig 2) [6,34].
Functionally, as revealed by ceca metatranscriptomics, mice susceptible to P. yoelii 17XNL hyperparasitemia had overexpression of filC, ureABC, and 6 members of nuo gene family related to gut microbes [36].Overexpression of filC, which encodes bacterial flagellin, is associated with mucosal barrier breakdown and inflammation [45,46].Short-chain fatty acids (SCFAs) are bacterial metabolites that have pleotropic effects on host health.SCFAs play a vital role in energy metabolism, immunologic homeostasis, and gut barrier integrity [47].Only the level of propionic acid (PA) among 7 other SCFAs was significantly higher in mice susceptible to P. yoelii 17XNL hyperparasitemia [39].Bacteroidetes, which are abundant in P. yoelii 17XNL hyperparasitemia susceptible mice [6], are able to ferment polysaccharides to PA [48].Bacteroides acidifaciens in the mouse gut are able to significantly increase the level of SCFAs especially PA [49].Higher level of PA in gut suppresses inflammation and ameliorates liver ischemia and reperfusion injury in mice.Increased levels of circulating PA is linked to higher cognitive decline in older persons [50] and associated with innate neuroinflammation, increased oxidative stress, glutathione depletion, and altered phospholipid/acylcarnitine profiles linked to autism spectrum disorder [48,51].The degree to which SCFAs contribute to malaria outcomes, including cerebral malaria, are unknown.
Plasmodium species infect red blood cells (RBCs) and undergo asexual replication to produce merozoites or enter sexual differentiation to produce gametocytes.It is not known if gut microbiota composition impact RBC physiology.Intriguingly, functional profiling of whole ceca (ceca content + ceca tissue) metatranscriptomics of P. yoelii 17XNL hyperparasitemiasusceptible mice revealed increased expression of basigin, a cell surface receptor required for P. falciparum invasion of RBCs [36].Recently, the gut bacteria Flavonifractor plautii was found to be involved in the conversion blood type A to universal O type blood [52].Higher abundance of Flavonifractor platutii was seen in the Ugandan kids with severe malaria [6].How gut bacteria impact the function of RBCs and Plasmodium biology within RBCs is an unexplored area.

Non-cerebral malaria models
While gut microbiota has been shown to impact malaria outcomes, research has also shown that Plasmodium infections can alter gut bacteria composition dependent on the murine model, parasite strain, malaria severity, and baseline gut microbiota.In C57BL/6 mice, P. yoelii nigeriensis infection increased gut Bacteroides and decreased Ruminococcus on day 10 post infection (Fig 2) [53].Bacterial diversities were lowest (alpha index) on day 10 p.i. and recovered to baseline level by day 30 post infection [53].A similar decrease in alpha diversity and increase in abundance of bacteria belonging to Bacteroidota (such as Alistipes and Bacteroides) were reported in BALB/c mice following lethal P. yoelii 17XL infection at day 5 post infection compared to baseline level (Fig 2) [54].In a nonhuman primate model, rhesus macaques infected by P. cynomolgi have decreased gut microbial alpha diversity at the peak of infection with a dramatic increase in relative abundance of Proteobacteria (family Helicobacteraceae) while decrease in Firmicutes (family Lactobacillaceae and Ruminococcaceae), Bacteroidetes (family Prevotellaceae) (Fig 2) [55].However, Denny and colleagues and Yawen and colleagues showed increase in alpha diversity in mice following P. yoelii 17XNL infection [38,56].Mice that are resistant to P. yoelii 17XNL hyperparasitemia (peak parasitemia is below 20%) have increase in alpha diversity (observed OTUs).In contrast, alpha diversity was stable in P. yoelii 17XNL hyperparasitemia-susceptible mice (peak parasitemia reaches up to 60%) following infection.Beta diversity (Bray-Curtis distance) was impacted by P. yoelii 17XNL infection in both hyperparasitemia-susceptible and -resistant mice, implying changes in predicted functional capacity [38].In contrast to a shift in gut microbiota composition during P. yoelii 17XNL infection, untargeted metabolomics showed modest alterations in metabolite profile of small intestine and ceca content and plasma during parasitemia except at peak parasitemia in both P. yoelii 17XNL hyperparasitemia-susceptible and -resistant mice [38].

Experimental cerebral malaria
Cerebral malaria is one of the most severe forms of malaria and a leading cause of malaria mortality in children (15% death rate) and adults (20% death rate) [57].Nearly a quarter of cerebral malaria survivors suffer from life-long neurological sequelae and ongoing comorbidities [58].Five studies have investigated the impact of experimental cerebral malaria (ECM) on gut microbiome composition in murine malaria [59][60][61][62] and one on upper gastrointestinal pathophysiology [63] with variable results.Taniguchi and colleagues (2015) and Shimada and colleagues (2019) reported intestinal pathology in C57BL/6 mice infected by P. berghei ANKA that develop ECM [62,63].ECM caused weight loss, multiple red gastric patches, detachment of epithelia, gastric gas retention, enlargement of goblet cells, small intestine shortening, increased intestinal permeability, and caused dysbiosis [63].Knowler and colleagues (2023) observed lengthening of small intestine in contrast to Shimada and colleagues (2019) [60,63].P. berghei ANKA infection in C57BL/6 mice caused changes in gut bacteria composition with increased abundance of class Clostridia; family Enterobacteriaceae, Verrucomicrobiaceae; and genus Akkermansia, Alistipes, and Alloprevotella and decrease Lactobacillaceae family (Fig 2) [59,60,62].Although P. berghei ANKA causes lethal ECM within 7 to 10 days, these P. berghei ANKA-induced changes in gut bacterial composition were long lasting if mice were treated with artemether [61].

Humans
There is limited information regarding the effect of malaria on gut microbiota composition in humans.Presently, 1 study has assessed this in the context of longitudinal analysis of stool bacteria compositions in Kenyan infants [64].In this study, stool samples from infants (n = 10) from birth to 10 months were collected roughly 2 weeks before and 2 weeks after a febrile malarial episode and artemether-lumefantrine treatment.In contrast to model organisms, measurements of gut microbiota composition using multiple metrics of alpha and beta diversity did not show significant difference in structure and composition of gut microbiota [64].That artemether-lumefantrine treatment had no effect on gut bacteria populations in the Kenyan infants, is consistent with a separate study in mice showing that artemether-lumefantrine and artesunate-amodiaquine treatment had no effect on gut bacteria communities [65].There are important limitations to this study that must be considered including the small sample size of 10 infants, narrow age range of participants (<10 months old), lack of severe malaria, and lack of higher-resolution sample collection whereby gut bacteria could have changed and reverted to baseline.Therefore, additional longitudinal studies in humans that address these limitations and expand the geographical representation of participants are warranted to gain deeper insight.As other inflammatory and severe diseases have been shown to change gut bacteria compositions [66][67][68], it would not be surprising if severe malaria in children is indeed associated with changes in gut bacteria compositions.

Implications of malaria-induced gut microbiota changes
These observations raise an important question, what effect does Plasmodium-induced changes in gut microbiota composition have on current/subsequent malaria outcomes?The one study to assess this question performed FMTs from convalescent P. yoelii 17XNL (day 60 post infection) hyperparasitemia-resistant and -susceptible mice into germ-free mice.As noted above, P. yoelii 17XNL hyperparasitemia-resistant and -susceptible mice showed changes in gut bacteria communities following P. yoelii 17XNL infection, with decreasing differences in gut bacteria communities between these groups of mice at convalescence compared to differences observed pre-P.yoelii 17XNL infection [38].Yet, the ex-germ-free mice colonized with gut microbiota from P. yoelii 17XNL hyperparasitemia-resistant and -susceptible mice were resistant and susceptible, respectively, to hyperparasitemia following P. yoelii 17XNL infection [38].These results demonstrate that, at least within this model, Plasmodiuminduced changes in gut bacteria composition do not change susceptibility to future Plasmodium infections.That P. yoelii 17XNL-induced changes in gut bacteria did not impact subsequent malaria outcomes is beneficial to human case-control studies, as it demonstrates the bacteria that are present prior to Plasmodium infection and cause susceptibility to severe malaria are not lost in the susceptible mice nor do these bacteria appear in the resistant mice.Therefore, even if P. falciparum causes changes in human gut bacteria populations, these data suggest that comparing children with asymptomatic P. falciparum infections compared to children with severe malaria has the potential to identify bacteria that contributed to the differential malaria outcomes.

Immunological mechanisms by which host microbiota can impact malaria outcomes
Host immunity is essential to control Plasmodium infection [69], but immune responses are also involved in the pathology of malaria [70][71][72][73].As such, it is crucial to understand the components of host immunity that both contribute to protection and pathogenesis in malaria and how these are regulated by gut microbiota.This knowledge has the potential to aid in the development of microbiome-based therapeutics to control Plasmodium infection and prevent life-threatening severe malaria.The contribution of innate and adaptive immunity during Plasmodium infection is reviewed elsewhere [74].Contribution of specific gut microbiome in immunity and immune-mediated disorders and CD4+ T cell differentiation and function is published previously [9,[75][76][77].Here, we have reviewed components of the immune system that contribute towards control and pathogenesis of malaria and discuss specific gut microbiota that might affect these components of host immunity.Whether and how gut-derived immune cells impact the gut-distal immune response to malaria and other infections is yet to be fully understood (Fig 3).

GC B cells and TFh cells
GC reactions are required for production of high-affinity antibodies and long-term memory against malaria [69,[78][79][80].We have shown that gut microbiota compositions dynamically regulate the quality and quantity of GC reactions during Plasmodium infection in C57BL/6 mice infected with P. yoelii 17XNL [6,34].Relatively higher abundance of Clostridium papyrosolven, Alistipes putredinis, Alistipes timonensis, and Lactobacillus reuteri at genus level and Bacteroidiaceae and Lacnospiraceae at family level were associated with susceptibility to hyperparasitemia while Lactobacillus johnsonii, among others, with resistance to hyperparasitemia correlating to GC responses in mice [6].Possibly, gut microbiota associated with resistance to hyperparasitemia might be linked to higher levels of SCFAs, flagellin, peptidoglycans, lipopolysaccharides, cross reactive epitopes, but not PA in the gut, resulting in B cells in Peyer's patches and the spleen producing better antibody responses [39,81,82].

Regulatory T cells
Foxp3+ Tregs have ubiquitous roles in anti-malarial immunity in both mice and humans, yet owing to the pleiotropic effect of these cells and the complexity of anti-malaria immunity, the full contribution of these cells to protection and pathology during malaria is not fully understood [69,87].Lower Treg cell numbers are associated with lower parasite burden and better outcomes in humans during blood-stage malaria [72,88,89].However, depletion of Treg cells in FoxP3-diphtheria toxin receptor (DTR) transgenic (DEREG) C57BL/6 mice did not decrease ECM severity suggesting a limited role in ECM [90].Still, depletion of Tregs or blocking Cytotoxic T-lymphocyte-Associated protein (CTLA-4) expressed on Tregs during a narrow window-of-time just prior to peak parasitemia enhanced immune responses and accelerated parasite clearance.This enhanced protection was attributed to increased Tfh:B cell interactions in GC reactions generating a more robust antibody response during blood-stage P. yoelii 17XNL infection in mice [72].Germ-free mice colonized with SFB have increased expression of RORγt + Tregs [91], and SCFAs (e.g., butyrate and propionate) produced by commensal gut bacteria promote peripheral Treg generation [92].Consistent with gut microbiota stimulating expansion of these cells, Tregs are significantly reduced in germ-free or antibiotic-treated mice [91].Although the mechanism is unknown, mice treated with antibiotics have significantly decreased P. yoelii 17XNL burden compared to untreated mice [6].

CD8+ T cells
Cytotoxic CD8+ T cells can protect against liver-stage malaria while providing little help in controlling blood-stage malaria [69,93].However, CD8+ T cells have been shown in rodent ECM to cause breakdown of the blood-brain barrier [94] and have been found in the brain microvasculature of humans [70,95,96].Several studies have demonstrated the ability of gut microbiota to regulate CD8+ T cell responses.Mice that develop more colitis-associated tumors have increased numbers of CD8+IFNγ+ T cells in lamina propria with higher relative abundance of Alistipes, Ruminococcus, Prevotellaceae, and lower abundance of Lachnospiraceae in the gut compared to mice that develop low tumor burden [97].In contrast, circulating numbers of gut-derived CD8+IFNγ+ T cells induced by a mixture of 11 gut bacterial species belonging to 8 genera (Bacteroides, Parabacteroides, Alistipes, Paraprevotella, Eubacterim, Ruminococcaceae, Phascolarctobacterium, and Fusobacterium) had enhanced anti-tumor immunity against subcutaneous engraftment of MC38 adenocarcinoma cell and anti-microbial immunity against oral Listeria monocytogenes challenge [98].The ability of gut microbiota to modulate CD8+ T cell responses during malaria, in particular during cerebral malaria, is an area ripe for exploration.

γδ T cells
γδ T cells recognize Plasmodium-infected erythrocytes and destroy infected RBCs either through cytotoxic molecules or antibody dependent phagocytosis [99,100].Additionally, liverstage dependent, low blood-stage Plasmodium parasite mass activates γδ T cells to produce IL-17, which protects mice from lethal cerebral malaria by promoting erythropoiesis [101].Administration of lactic acid producing Lactobacillus plantarum probiotic promotes hematopoiesis and erythropoiesis [102], whether lactic acid-producing bacteria confer protection against severe malaria remains unclear.Interestingly, C57BL/6 mice fed a high fat diet (HFD) are resistant to ECM [103].Numerous factors may contribute to HFD protection to ECM, but a HFD can modulate gut microbiota, generally leading to a decrease in Bacteroidetes and increase in Firmicutes and Proteobacteria [104].Mice fed HFD for 3 weeks have increased number of IL-17+ γδ T cells, IFNγ+ Th1 cells, and CD8+ T cells while decreased numbers of Tregs in the colon and small intestine [105].However, increased abundance of Proteobacteria and lower abundance of Clostridiaceae and S24-7 is also associated with induction of CD4 + Tregs cells [106], highlighting the complex interaction between gut microbiota and immune system.Whether HFD protection from ECM is attributed to diet-dependent effects on gut microbiota and their modulation of host immune cells or alternative microbiome-independent effects is not known.

NKT cells
NKT cells have been shown to reduce blood-stage parasitemia due to enhanced secretion of IFNγ [107].NKT cells are usually found in thymus, spleen, liver, and bone marrow [108].Depleting gram-positive gut bacteria with vancomycin that are involved in the conversion of primary bile acids to secondary bile acids was able to induce hepatic NKT cell accumulation and decreased liver tumor growth [109].Vancomycin-treated mice had negligible presence of Bacteroidales and significantly reduced Clostridiales [109].Interestingly, monocolonization with Clostridium scindens, which can transform bile acid, was able to reduce hepatic NKT cells and recover Bacteroidales [109,110].Yet, the impact of gut microbiota modulating NKT cells other than in the liver is unknown; therefore, the ability of gut microbiota to modulate NKT cells and impact blood-stage parasite burden is unknown.Presently, there are no reported effects of gut microbiota on Plasmodium liver-stage burden, but as NKT cells are believed to have a protective role in liver-stage Plasmodium infections [74,111], it raises the possibility gut microbiota may impact liver-stage burden via modulation of NKT cells.

Natural killer (NK) cells
NK cells have beneficial roles during Plasmodium infection [112].NK cells produce inflammatory cytokines, kill infected RBCs, and participate in initiation and development of adaptive immune response during malaria infection [113][114][115][116]. Butyrate is one of the major SCFAs produced by gut microbiota that is reported to limit the effector function of human NK cells from blood in vitro by down-regulation of mTORC1 activity, c-Myc mRNA expression, and metabolism [117].In contrast, dietary butyrate supplementation or treatment with Clostridium butyricum in mice treated with antibiotics early in life promoted the maturation and restored function of liver-resident NK cells [118].Faecalibacterium, Roseburia, Fusobacteria, and Eubacterium are other bacteria that can produce butyrate [119].Additionally, high salt diet can enhance NK cell functions in a gut microbiota dependent way by increasing the abundance of probiotic bacteria Bifidobacterium but increased gut permeability [120].

Monocytes
Monocytes have both protective and pathological role in malaria infection [121].Enhanced p53 expression in monocytes are associated with attenuated Plasmodium-induced inflammation and protects from early fever during malaria infection in humans [30].IFNγ leads to enhanced expression of p53 in monocytes to attenuate pro-inflammatory activation [30,122,123].Gut microbial products have been associated with regulation and function of splenic monocytes [124].Oral antibiotics treatment eliminated bacterial taxa from Bacteroides and Firmicutes along with other bacteria that reduced pattern recognition receptor ligands in the serum that led to immature phenotype of splenic Ly6C high monocytes exhibiting decreased level of pro-inflammatory cytokines and increased phagocytic abilities [124].

Targeting gut microbiota as a strategy to decrease severe malaria
There are numerous approaches by which gut microbiota can be modulated.In this section, we have reviewed some of the past efforts and recent advances and strategies to manipulate gut microbiota.These approaches may serve as useful approaches to interrogate gut microbiotahost-parasite interactions, with some approaches serving as potential gut microbiota-based approaches to mitigate severe malaria.The latter will require extensive investigation and clinical trials with positive malaria outcome before any recommendations can be made (Fig 3).

High fat, calorie restricted, and low protein diets
Studies have shown that high fat, calorie restricted, and low protein diets are associated with favorable parasitemia and mortality outcomes in rodent malaria models [103,125,126].However, the role of gut microbiota, mechanisms of action, and identification of targetable pathways are required to advance dietary gut microbiota-based therapeutics.

Milk
Rodents on a milk diet suppress P. berghei infection, while having no effect on Nutallia rodhaini (Babesia) or Trypanosoma brucei infection [127][128][129].Similar protections were seen against 2 nonhuman primate (monkey) strains, P. knowlesi and P. cynomolgi [129].Moreover, mice and monkeys fed a milk diet supplemented with p-aminobenzoate, a growth factor for many Plasmodium species, lost protection against malaria [129].Milk consumption has been shown to decrease Bacteroidetes and Prevotella while increase Proteobacteria, Bifidobacterium, Lactobacillus, and Roseburia [130,131].Connections between milk consumption and its effect on gut microbiota to modulate malaria severity warrants further investigation.

Folic acid (FA)
Controlled trials have found that FA supplementation compromised the efficacy of antimalaria drugs and should be avoided as supplement in children in malaria endemic regions [132,133].FA induces Tregs [134] and production of folate is positively associated with higher relative abundance of Bacteroides, Sutterella, and Parasutterella [135] that are associated with high blood-stage parasite burden.

Probiotics
Probiotics are valued for the health benefits they confer upon the host, and these have been investigated in the context of Plasmodium infections.Heat killed Lactobacillus sakei HS-1 was able to minimize weight loss and mitigate intestinal pathology and limited small intestine shortening in C57BL/6 mice during P. berghei infection [136].However, parasitemia load was not decreased in Lactobacillus sakei-treated mice [136].Likewise, L. casei administration was shown to be protective against P. berghei as an adjunct therapy along with antimalarial treatment in BALB/C mice [137,138].Additionally, C57BL/6 mice administered with Bifidobacterium longum alone had diminished P. berghei burden, colon inflammation, and significantly lower level of plasma TNFα and IFNγ compared to B. longum plus L. casei or L. casei alone [139].Expression of intestinal CD103+ dendritic cells and intestinal Tregs were high in mice receiving B. longum [139].CD103+ dendritic cells in nonlymphoid organs induces Tregs [140].Dendritic cells have a critical role in initiating and regulating innate and adaptive immunity against malaria [141].Finally, some probiotic bacteria strains express high levels of α-gal [142], which may facilitate protection against Plasmodium exoerythrocytic stages via induction of anti-α-gal antibodies, as discussed above.
One of the challenges with probiotics is poor engraftment.Understanding the dietary requirements of probiotic bacteria will be important as it can be targeted to improve engraftment.For example, some strains of Bacteroides ovatus can utilize the marine polysaccharide, porphyran, which is lacking in the vast majority of gut bacteria [143].Transfer of the gene cluster for porphyran utilization from B. ovatus into another Bacteroides species lacking this cluster allowed fine tuning the engraftment of these bacteria into the competitive mouse gut microbiota niche when mice were provided porphyran in their diet.Similar approaches can be used to clone unique nutrient utilization gene clusters into probiotic strains with proven beneficial effect against malaria to overcome the probiotic wash-out effect.

Antimicrobial peptides
Non-immunogenic and non-toxic antimicrobial peptides from Lactobacillus plantarum strain LR/14 inhibited the growth of P. falciparum in vitro without any hemolysis [144].L. plantarum can also inhibit many gram-positive and gram-negative bacteria [145], which can be exploited to manipulate gut microbiota composition.

Antibiotics
C57BL/6 mice treated with any of 4 antibiotics (ampicillin, gentamicin, metronidazole, and vancomycin) in drinking water prior to and during P. yoelii 17XNL infection significantly decrease the parasite burden.Of note, treatment of mice resistant to P. yoelii 17XNL hyperparasitemia showed no effect (i.e., the mice did not become susceptible).Moreover, treating with oral vancomycin 1 week prior to P. yoelii 17XNL infection, followed by cessation of treatment, significantly decreased P. yoelii 17XNL parasite burden.Intriguingly, preinfection vancomycin treatment afforded resistance to P. yoelii 17XNL hyperparasitemia for at least 3 months postcessation of vancomycin treatment.Although antibiotic use poses threat to emergence of antibiotic resistance, clinical trials performed with antibiotics (amoxicillin, cefdinir, ceftriaxone, metronidazole) were associated with positive outcome in malnourished children [146][147][148].Thus, clinical trials to test antibiotics as an adjunct therapy to prevent and manage severe malaria may have merit.

Bacteriophages
Bacteriophages are viruses that can attack bacteria with specificity [149].Bacteriophages, natural or engineered, can dynamically modulate gut microbiota and the metabolome [150].Bacteriophages have been used to precisely modulate gut microbiota in diseases like IBD, colitis, type 2 diabetes, and colorectal cancer among others to improve prognosis with minimal damage to host gut microbiota [149,151,152].Of course, this exciting technology comes with limitations and challenges [149].For example, evolution of phage resistance, efficacy of phages against biofilms, and stability of phage preparations are a few important challenges [153].Nevertheless, if specific gut bacteria are involved in susceptibility to severe malaria, then targeting these bacteria via bacteriophages might result in promising outcomes.

Short chain fatty acids (SCFAs)
Previously, we have shown that levels of SCFAs like propionic acid, butyric acid, and valeric acid were significantly different among mice susceptible and resistant to P. yoelii 17XNL hyperparasitemia [39].Culminating evidence suggests important roles of SCFAs in the development and regulation of the immune system and gut barrier integrity that impact disease severity and enhanced health [154,155].Consequently, SCFAs may impact malaria severity; however, the contribution of any specific SCFAs in malaria severity is yet to be studied.

Dietary tryptophan and indoles
Gut microbiota conversion of dietary tryptophan to indoles has important roles in enhancing gut barrier integrity and binds to AhR receptor on immune cells to induce anti-inflammatory and antimicrobial properties [156,157].The indole moiety is one of the most promising chemotypes for the development of antiparasitic drugs [158].Indole-3-acetic acid activated AhR pathway promotes anti-inflammatory cytokine IL-10 and up-regulated Foxp3 and increased Treg cells in a proteoglycan (PG)-induced ankylosis mouse model [159,160].Therefore, the role of gut-derived indoles and the ability of indoles to induce Tregs in malaria will be an interesting avenue of research [161,162].

Conclusion
Within the last 2 decades, our understanding of gut microbiota function has increased exponentially due to advances in high-throughput omics technologies like nucleic acid sequencing, metabolomics, metatranscriptomics, proteomics, interbacterial and intrabacterial interaction within the host, interdisciplinary studies, and the rise of artificial intelligence and machine learning in biological sciences.With this new knowledge, the scientific community is positioned to identify specific gut microbiota and microbiota-derived products and their interactions with the host immune system in modulating the severity of malaria.The capacity to precisely identify these biomarkers will increase with continued development and innovation in the microbiome field.With these advancements, gut microbiome-based therapies may one day be used to mitigate the severity of malaria associated with Plasmodium infection.

Fig 1 .
Fig 1.Gut bacteria associated with human malaria.To date 5 peer-reviewed studies have been published on the impact of the gut microbiota in Plasmodium infection in humans.A cohort of children in Kalifabougou, Mali was used for 2 prospective studies (Yilmaz and colleagues (2014)[18] and Yooseph and colleagues (2015)[28]).Additionally, 3 case-control studies investigated association of gut microbiota at the time of Plasmodium infection (Huwe and colleagues (2019)[32] and Easton and colleagues (2020)[33]) and SMA (Mandal and colleagues (2021)[6]).Prospective and case-control studies have their own advantages and disadvantages.Bacteria associated with the clinical outcome is shown.Pf: P. falciparum, neg: negative, pos: positive.Figure was created with BioRender.com.
Fig 1.Gut bacteria associated with human malaria.To date 5 peer-reviewed studies have been published on the impact of the gut microbiota in Plasmodium infection in humans.A cohort of children in Kalifabougou, Mali was used for 2 prospective studies (Yilmaz and colleagues (2014)[18] and Yooseph and colleagues (2015)[28]).Additionally, 3 case-control studies investigated association of gut microbiota at the time of Plasmodium infection (Huwe and colleagues (2019)[32] and Easton and colleagues (2020)[33]) and SMA (Mandal and colleagues (2021)[6]).Prospective and case-control studies have their own advantages and disadvantages.Bacteria associated with the clinical outcome is shown.Pf: P. falciparum, neg: negative, pos: positive.Figure was created with BioRender.com.https://doi.org/10.1371/journal.ppat.1011665.g001

Fig 2 .
Fig 2. Gut bacteria associated with nonhuman model of malaria.Top 2 panels show baseline gut bacteria differentially abundant in mice either susceptible or resistant to P. yoelii 17XNL hyperparasitemia and P. chabaudi AS pregnancy outcomes.Fecal pellets are collected at baseline prior to Plasmodium infection to determine the gut microbiota composition.Bottom 2 panels show the shift in gut microbiota composition during Plasmodium infection in mice and monkeys.In nonlethal infection models, fecal pellet microbiota composition at peak parasitemia is compared to before Plasmodium infection.Bacteria that are significantly increased (up arrow) or decreased (down arrow) are shown.P. yoelii 17XL causes lethal infection due to hyperparasitemia while P. berghei ANKA causes mortality due to experimental cerebral malaria.Gut fecal samples are collected before mortality and compared to baseline gut microbiota.Changes in bacteria population at phylum, family, and genus level are shown.Figure was created with BioRender.com.https://doi.org/10.1371/journal.ppat.1011665.g002

Fig 3 .
Fig 3. Gut microbiota intervention strategy and possible immunological mechanism of action against severity to malaria.Agents that can modulate gut microbiota composition or deplete gut bacteria that can influence gut or systemic immunity are shown.Dotted arrow indicates the potential interactions which need further validation.Color of arrows connect gut microbiota modifying agents and their impact on respective immune cells.Role of different immune populations to inhibit or exacerbate various stage and types of malaria are connected.Although, the exact mechanism on how gut microbiota impacts severe malaria is unknown, this figure provides a plausible connection between gut microbiota and malaria severity.Figure was created with BioRender.com.https://doi.org/10.1371/journal.ppat.1011665.g003